Schäfer, N., Maierhofer, T., Herrmann, J., Jørgensen, M. E., Lind, C., vonMeyer, K., ... Hedrich, R. (2018). A Tandem Amino Acid Residue Motif inGuard Cell SLAC1 Anion Channel of Grasses Allows for the Control ofStomatal Aperture by Nitrate. Current Biology, 28(9), 1370-1379.e5.https://doi.org/10.1016/j.cub.2018.03.027

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Schäfer et al.

1

A tandem amino acid residue motif in guard cell SLAC1 anion channel of grasses allows 1

for the control of stomatal aperture by nitrate 2

3

Nadine Schäfer1†, Tobias Maierhofer1†, Johannes Herrmann1†, Morten Egevang Jørgensen1, 4

Christof Lind1, Katharina von Meyer1, Silke Lautner2, Jörg Fromm2, Marius Felder3, Alistair 5

M. Hetherington4*, Peter Ache1, Dietmar Geiger1*, Rainer Hedrich1* 6

7

1 Institute for Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, 8

Biocenter, University of Wuerzburg, Julius-von-Sachs Platz 2, D-97082 Wuerzburg, Germany 9

2 Department of Wood Science, University Hamburg, Leuschnerstr. 91d, D-21031 Hamburg, 10

Germany 11

3 Plant Genome and Systems Biology, Helmholtz Center Munich, Ingolstädter Landstr. 1, D-12

85764 Neuherberg, Germany 13

4 School of Biological Sciences, Life Sciences Building, University of Bristol, 24 Tyndall 14

Avenue, Bristol BS8 1TQ, UK. 15

† These authors contributed equally. 16

*To whom correspondence should be addressed: 17

Rainer Hedrich, Email: hedrich@botanik.uni-wuerzburg.de, Tel.: +49 (0)931 318 6100 18

Alistair Hetherington, Email: alistair.hetherington@bristol.ac.uk, Tel.: +44 (0) 117 39 41188 19

and Dietmar Geiger (Lead Contact), Email: geiger@botanik.uni-wuerzburg.de, Tel.: +49 20

(0)931 318 6105 21

Field Code Changed

Field Code Changed

Schäfer et al.

2

Abstract 22

The latest major group of plants to evolve were the grasses. These became important in the mid-23

Paleogene about 40 million years ago. During evolution leaf CO2 uptake and transpirational 24

water loss were optimized by the acquisition of grass specific stomatal complexes. In contrast 25

to the kidney-shaped guard cells (GCs) typical of the dicots such as Arabidopsis, in the grasses 26

and agronomically important cereals, the guard cells are dumbbell-shaped and are associated 27

with morphologically distinct subsidiary cells (SCs). We studied the molecular basis of guard 28

cell action in the major cereal crop barley. Upon feeding ABA to xylem sap of an intact barley 29

leaf, stomata closed in a nitrate dependent manner. This process was initiated by activation of 30

guard cell SLAC-type anion channel currents. HvSLAC1 expressed in Xenopus oocytes gave 31

rise to S-type anion currents that increased several fold upon stimulation with >3 mM nitrate. 32

We identified a tandem amino acid residue motif that within the SLAC1 channels differs 33

fundamentally between monocots and dicots. When the motif of nitrate-insensitive dicot 34

Arabidopsis SLAC1 was replaced by the monocot signature, AtSLAC1 converted into a grass-35

type like nitrate-sensitive channel. Our work reveals a fundamental difference between monocot 36

and dicot guard cells and prompts questions into the selective pressures during evolution that 37

resulted in fundamental changes in the regulation of SLAC1 function. 38

Schäfer et al.

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Introduction 39

Guard cell pairs that drive stomatal movement control leaf CO2 uptake and concomitant 40

transpirational water loss. To survive episodes of drought and excessive heat, stomata have to 41

sense sudden changes in environmental conditions and adjust stomatal aperture accordingly. 42

Based largely on work in the model dicot Arabidopsis much is known about the molecular 43

details that underlie stomatal function [1-3]. By way of contrast, in the grasses that are by far 44

the world's most important sources of food, it is surprising that our molecular knowledge 45

concerning guard cell function is still very limited [4-8] (and references therein). 46

In contrast to the kidney-shaped guard cells typical of the dicots, grass stomata come as pairs 47

of dumbbell-shaped guard cells (GCs) in physical contact with a lateral pair of subsidiary cells 48

(SCs, [4]). In terms of function there Raschke and Fellows (1971, [9]) found that during 49

stomatal opening in maize K+ and anions are shuttled from SCs to GCs and when closing they 50

move in the opposite direction. Guard cells of dicots and monocots respond to the plant 51

hormone ABA, that binds to its cytosolic PYR/PYL/RCAR type receptor [10, 11]. The ABA 52

receptor forms a complex with the PP2C phosphatase ABI1 and SnRK2 kinase OST1 [12]. 53

Binding of ABA to the receptor ABA cause inactivation of ABI1, and release of OST1 from 54

inhibition of the PP2C phosphatase [13]. In turn, the OST1 kinase phosphorylates the guard cell 55

anion channel SLAC1, causing it to open [14, 15]. The resulting release of anions depolarizes 56

the plasma membrane and the change in voltage activates the GORK1 channel resulting in K+ 57

efflux, which is followed by the loss of water and resulting stomatal closure [1, 16]. 58

Potassium and chloride represent the dominant ions in dicot and monocot guard cells. In the 59

dicot model plant Arabidopsis, the SLAC1 channel is permeable to chloride. When expressed 60

in the heterologous expression system of Xenopus oocytes, Arabidopsis SLAC1 is active in 61

chloride-based media [14]. In contrast to SLAC1, its homologs SLAH2 and 3 require nitrate at 62

the external mouth of the anion channel to gate open in oocyte and guard cell systems [17, 18]. 63

In order to gain insights into how cereal guard cells function, we analysed the transcript profile 64

of barley guard cells and subsidiary cells. We identified HvSLAC1 together with the major 65

components of guard cell ABA signalling. Strikingly, in barley SLAC1 and in marked contrast 66

to Arabidopsis SLAC1, we identified a distinct tandem amino acid motif, responsible for nitrate 67

activation. 68

Schäfer et al.

4

Results 69

ABA-dependent stomatal closure in barley requires extracellular nitrate 70

Surprisingly, studies aimed at understanding the molecular basis of stomatal movements in 71

barley, a major cereal grass crop, are somewhat limited [4, 5]. To address this issue, we first 72

investigated stomatal function in the intact barley leaf using IRGA-based measurements of 73

stomatal conductance. We focussed on Hordeum vulgare line Barke, because genome 74

information is available, and it represents one of the most popular brewing barley varieties 75

worldwide. 76

From work in Arabidopsis it is known that ABA triggers stomatal closure following binding to 77

cytosolic receptors of the PYR/PYL-family and the subsequent induction of a signalling 78

pathway that ultimately activates SLAC/SLAH anion channels [14, 15, 17, 19, 20]. To 79

investigate the response of barley stomata to ABA, we fed 25µM ABA (a concentration 80

sufficient to close barley stomata [5]) via the leaf petiole. When the petiole was supplied with 81

ABA in water stomatal closure was delayed and incomplete (figure 1A). This is in marked 82

contrast to the prompt closure observed in the dicot Arabidopsis [21], but is similar to the 83

response observed in the monocot date palm [21]. However, the most striking result to emerge 84

from this experiment was that timely and significant ABA-induced stomatal closure could be 85

elicited by the addition of nitrate (5 mM KNO3-) to the feeding medium (figure 1B). This results 86

clearly shows that in barley the full stomatal response to ABA requires the presence of nitrate. 87

88

Stomatal closure in barley involves inverse fluxes of K+ and Cl- between guard and 89

subsidiary cells 90

Having identified a requirement for nitrate in ABA-induced closure we next investigated the 91

requirement for K+ and Cl-. Given that early studies in maize provided initial evidence for K+ 92

and chloride shuttling between guard cells and subsidiary cells [9, 22], we used EDX analysis 93

to determine the content of these elements in guard and subsidiary cells. Figure 1C shows that 94

guard cells (GC) from closed stomata contained less K and Cl than open ones. Subsidiary cells 95

(SC), however, exhibited the inverse relationship with relatively higher levels of K and Cl 96

associated with closed stomata (figure 1C). These data confirm older work in maize [9, 22] but 97

leave open the question of why there is a requirement for nitrate for the closure of barley 98

stomata. In summary, in barley just as in Arabidopsis, K+ and Cl- represent major ionic 99

components of the guard cell osmotic motor that drives stomatal movement. The fact that the 100

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5

changes in K+ are larger than those of Cl-, indicate that besides Cl-, additional anions such as 101

nitrate or malate must be involved. In this context, it should be mentioned that in barley 102

epidermal cells Cl- and NO3- might provide the total negative solute charge [23]. 103

104

Using transcriptomics to investigate ABA-induced stomatal closure in barley 105

To investigate the molecular machinery responsible for bringing about stomatal closure in 106

barley and in particular the ion fluxes between guard and subsidiary cells, we employed a 107

transcriptomic approach. We isolated three experimental preparations; 1) intact leaves (L); 2) 108

lower epidermis with intact stomatal complexes (GCSC) and 3) leaves without upper and lower 109

epidermis und thus without guard cell complexes (LwoGC). We employed a bioinformatic 110

approach to identify differentially expressed genes (DEG) in guard cell complexes (for full 111

DEG list see table S1). Because guard cells, in contrast to mesophyll cells, contain a reduced 112

number of chloroplasts and epidermal pavement cells have no chloroplasts, we initially 113

compared photosynthesis (PS) related transcripts. As expected, we found genes associated with 114

PS electron transport and CO2 fixation underrepresented in the GCSC samples (table S1, filtered 115

by MapMan category 1). When analysing RNA-seq data, it did not come as a surprise that we 116

found DEGs related to guard cell function- and ABA signalling. 117

We next investigated genes involved in ion transport. In the ion channel fraction guard cell-118

specific anion channels of the SLAC/SLAH type and Shaker-like potassium channels of the 119

KAT- and GORK/SKOR-type were identified (table S2). This expression pattern underpins the 120

notion that the grass type stomata from barley harbour the guard cell major H+, K+ and Cl- 121

transporting entities represented by AHA1 and KAT1 for stomatal opening [24] and SLAC1 and 122

GORK for closure ([1] for review). Among the ABA dependent transcripts, pronounced guard 123

cell expression of orthologues to the PYR/PYL ABA receptor family, PP2C phosphatases ABI1 124

and 2, SnRK2 kinases of the OST1-type were found. Based on 5 HvOST1-like sequences, we 125

generated a phylogenetic tree and identified HvOST1.1, 1.2 and 1.3 most closely related to the 126

Arabidopsis OST1 (figure S1A). 127

To validate expression of the guard cell ion channels and components of the ABA signalling 128

pathway suggested by the RNA-seq data, we performed qPCR analysis. We sampled leaves (L) 129

and epidermal peels containing both guard cells and subsidiary cells (GCSC) and preparations 130

in which the subsidiary cells were selectively disrupted using the blender method [25]. The 131

latter preparation represented a fraction highly enriched in guard cells (GC) (figure S1B). As in 132

Arabidopsis guard cell databases obtained using a related experimental approach [25], we found 133

Schäfer et al.

6

transcripts of ABI1, SLAC1 and OST1.1 expressed in an almost guard cell specific manner. In 134

contrast, OST1.3 expression was found distributed equally among samples. Among the guard 135

cell expressed SnRK2 genes, HvOST1.1 shows the closest phylogenetic relationship to the 136

Arabidopsis OST1 kinase with 80.5% identical residues (figure S1A). The HvOST1.1 sequence 137

harbours all known functional domains of OST1-like, ABA-dependent SnRK2 kinases, 138

including the DI domain/SnRK2 box and the DII domain/ABA-Box [12, 26-28]. On these 139

grounds HvOST1.1 very likely represents the SLAC1 activating ABA kinase in barley. In 140

Arabidopsis GCs SLAH3 operates a nitrate activated anion channel conducting nitrate and 141

chloride [17]. In barley, however, SLAH3.1 was found expressed in leaves, but not in the GCSC 142

and GC samples (figure S1B). 143

144

Barley HvSLAC1 is under the control of nitrate and OST1 /ABI1 pair 145

McAdam et al., 2016 [29] and Lind et al., 2015 [30] showed that SnRK2 kinases (OST1s) are 146

strictly conserved during evolution. All OST1 kinases derived from different evolutionary 147

distinct plant species so far tested are capable of activating Arabidopsis SLAC1. In addition, 148

AtOST1 is capable of activating SLAC1 isoforms from other monocot species, such as 149

PdSLAC1 from date palm [21] or OsSLAC1 from rice [8]. Thus, to understand the molecular 150

basis of nitrate dependency of stomatal closure in barley, we expressed HvSLAC1 alone and 151

together with AtOST1 in Xenopus oocytes and studied its anion channel properties. In the 152

absence of the SnRK2 kinase no currents were recorded and even in the presence of the ABA-153

induced kinase AtOST1 and 30 mM chloride-based extracellular media, macroscopic S-type 154

anion currents could only be recorded at strongly depolarized membrane potentials (figure 2A). 155

Upon addition of 30 mM nitrate, however, pronounced S-type anion currents were observed 156

(figure 2A). While the Arabidopsis OST1 WT kinase was capable of activating HvSLAC1 in 157

nitrate-based solutions, the kinase dead mutant AtOST1 D140A could not perform this function 158

(figure 2B and C). This indicates that phosphorylation of HvSLAC1 is strictly required for 159

anion channel activation (cf. [14]). Besides the calcium independent SLAC1 kinase OST1, 160

calcium dependent kinases of the CPK and CIPK/CBL type can phosphorylate and gate open 161

AtSLAC1 ([31]). As a representative of the latter kinases category, we selected CPK6 for 162

oocyte co-expression experiments with HvSLAC1. As with OST1, CKP6 activated the barely 163

S-type channel to the same extent as AtSLAC1 (figure 2A; figure S2A; [19, 32]). 164

In contrast to the grass SLAC1, Arabidopsis SLAC1 does not require the presence of 165

extracellular nitrate for activation (figure S2A, c.f. [18]). Interestingly, the ABA phosphatase 166

Schäfer et al.

7

ABI1 in Arabidopsis guard cell inhibits the response to nitrate [33] and negatively regulates 167

AtOST1 and AtSLAC1 activity [14, 15, 19, 31]. Upon co-expression of HvSLAC1 and AtOST1 168

with ABI1, we found that HvSLAC1-mediated anion currents were strongly reduced (figure 2B 169

and C). These findings indicate that fast ABA signalling is conserved between guard cells of 170

the dicot Arabidopsis and monocot grass Hordeum vulgare. 171

The Arabidopsis AtSLAC1 does not require extracellular nitrate for activation (figure S2A) but 172

has a strong permeably preference for nitrate over chloride [14]. The HvSLAC1 PNO3/PCl 173

calculated permeability ratio of 6.4 ± 0.8 indicates that the dicot and monocot guard cell anion 174

channel would preferentially conduct nitrate when present in guard cells at osmotically relevant 175

quantities (figure S2B). Is the nitrate sensitivity a unique feature of HvSLAC1 or is it found in 176

other cereals, too? To answer this question we expressed rice SLAC1 (OsSLAC1) in oocytes 177

(cf. [8]). The results in figure S2A and C show that the rice S-type anion channel shared its 178

selectivity and nitrate dependent features with the Hordeum SLAC1 anion channel. In this 179

context, it should be noted that we recently showed, in the monocot Phoenix dactylifera (date 180

palm), that PdSLAC1 is also nitrate activated [21]. Besides nitrate, other physiological relevant 181

anions such as phosphate, sulphate, malate or chloride were not capable of activating 182

HvSLAC1-derived anion currents (figure S2D) or to shift its rel. PO to negative (physiological) 183

membrane potentials (figure S2E). 184

To find out whether nitrate activation is a property of monocot SLAC1, we compared the 185

Brassicaceaen AtSLAC1 with a dicot orthologue from tomato and tobacco - two Solanaceae 186

crop species. These dicot SLAC1s behave more similarly to the nitrate insensitive AtSLAC1 187

than to nitrate-activated monocot SLAC1s (figure 2D). 188

189

Extracellular nitrate primes HvSLAC1 to release chloride 190

To study the biophysical properties of nitrate-activated HvSLAC1 in more detail we co-191

expressed the anion channel with AtCPK6 [2, 19, 32] and determined current densities and the 192

relative open probability as a function of the external nitrate concentration (figure 3A and B). 193

When exposed to increasing external nitrate concentrations, the peak efflux currents and the 194

relative open probability shifted towards negative membrane potentials and thereby increased 195

the plasma membrane anion conductance (figure 3A and B). In contrast, similar experiments 196

with increasing external chloride applications revealed that in the physiological membrane 197

potential range (negative from -100 mV), anion release currents are absent irrespective of the 198

external Cl- concentration (figure 3C and D). While 100 mM nitrate shifted the HvSLAC1 half-199

Schäfer et al.

8

maximal open probability (V1/2) to -120 mV (figure 3B), V1/2 in 100 mM chloride remained at 200

depolarized membrane potentials of -20 mV (figure 3D). When plotting V1/2 as a function of 201

the external nitrate concentration, the resulting saturation curve could be described with a 202

Michaelis-Menten equation (figure 3E) resulting in a K0.5 value of 10.9 ± 3.8 mM nitrate. Thus 203

physiological [NO3−] concentration of 10 to 70 mM found in the xylem sap of barley leaves 204

[34, 35] will activate HvSLAC1 anion channel. This set of experiments shows not only that 205

HvSLAC1 conducts nitrate (figure 3A and B, figure S2B) but also that nitrate is required to 206

gate the barley guard cell channel open. 207

Nitrate-dependent gating is a known feature of SLAH2 and SLAH3 branch of the Arabidopsis 208

SLAC/SLAH anion channel family, but not of its founding member AtSLAC1 (see above and 209

c.f. [17, 18]; figure S2A). While AtSLAH2 is strictly nitrate selective, AtSLAH3 also conducts 210

chloride when primed with extracellular nitrate [17, 18]. To further examine the nature of the 211

nitrate dependency of HvSLAC1 and to substantiate that HvSLAC1 conducts chloride in the 212

presence of its gating ligand nitrate, the anion channel was challenged by different chloride to 213

nitrate ratios. Anion currents recorded in the presence of 3 mM extracellular chloride were very 214

weak and reversed at +50 mV (figure 3F). In contrast, addition of 3 mM nitrate enhanced the 215

steady state currents and shifted the reversal potential to 0 mV (figure 3F). When the chloride 216

concentration was further increased to 100 mM in the presence of 3 mM nitrate, the reversal 217

potential of HvSLAC1-mediated anion currents shifted to more negative membrane potentials 218

without anion release currents and relative open probability being influenced by chloride (figure 219

3F and G). To further investigate the chloride conductance of HvSLAC1 when primed with 220

extracellular nitrate, we monitored the reversal potential of HvSLAC1 AtCPK6 expressing 221

oocytes. Upon addition of 3 mM nitrate to a 3 mM chloride containing bath solution, the 222

reversal potential dropped by 56 mV and shifted to even more negative membrane potentials 223

when the chloride concentration was increased to 100 mM (figure S2F and G). This behaviour 224

and those to varying Cl- to NO3- rations (figure 3F and G) indicate that HvSLAC1, when pre-225

activated by nitrate, conducts both nitrate and chloride. In contrast to the nitrate sensitive 226

monocot S-type anion channels, AtSLAC1 reversal potential shifts appeared less nitrate- but 227

more chloride sensitive (figure S2F and G). In contrast to AtSLAC1, OsSLAC1 and HvSLAC1, 228

the reversal potential of the nitrate-selective AtSLAH2 was sensitive to nitrate only but not 229

chloride (figure S2F and G). Taken together, the electrical properties of the monocot anion 230

channels are reminiscent of the nitrate-gated, chloride and nitrate permeable AtSLAH3 anion 231

channel rather than of the nitrate-independent dicot AtSLAC1. 232

Schäfer et al.

9

233

SLAC1 grass type tandem amino acid motif on TMD3 is key for nitrate priming 234

3D homology modelling of AtSLAC1 and AtSLAH2 to the crystal structure obtained with the 235

bacterial homologue HiTehA in combination with site-directed mutagenesis showed associated 236

residues of Trans-Membrane Domain TMD3 as part of the pore forming entity [18, 36]. To find 237

the nitrate site in barley SLAC1, we compared TMD3 of monocot and dicot SLAC1 type 238

channels (figure 4A, figure S3). Monocot and dicot SLAC1s could be well distinguished by 239

two residues close to Val272 and Val273 of AtSLAC1. We found dicot SLAC1s to either carry 240

two valine residues such as AtSLAC1 (V272 and V273) or an IV pair in Solanaceaen species. 241

Monocots including barley HvSLAC1 and date palm PdSLAC1 at the related positions harbour 242

an isoleucine and alanine side chain (figure 4A, Fig S3) similar to the nitrate activated SLAH2/3 243

anion channels that possess either the IA or an IS motif (figure S3). Given that the amino acid 244

sequence on TMD3 clearly distinguishes monocot from dicot SLACs, these residues seem to 245

represent a specific signature. Thus, we tested whether this TMD3 tandem motif between both 246

monocot and dicot representative SLACs is essential for nitrate dependency. Therefore, we 247

replaced just the VV motif in AtSLAC1 by IA and the IA motif in Hv/PdSLAC1 by the dicot 248

VV motif. The resulting Arabidopsis mutant AtSLAC1 V272I V273A displayed nitrate-249

induced anion currents just like HvSLAC1 and PdSLAC1 WT (figure 4B). Note, this behaviour 250

appeared only in 30 % of the tested oocyte batches (this conditional phenotype is shown in 251

figure 4B) whereas the remaining oocyte batches revealed a AtSLAC1 WT behaviour. Thus, 252

the introduction of the monocot IA motif in TMD3 of AtSLAC1 is essential and sufficient to 253

provide for the nitrate dependency. However, when the monocot SLACs were equipped with 254

the dicot VV signature the resulting mutants with barley (HvSLAC1 I286V A287V) and date 255

palm anion channel (PdSLAC1 I285V A286V), appeared severely impaired even in nitrate-256

based buffers (figure 4B) indicating that in monocots additional structural moieties shape the 257

permeation pathway. The fact that the HvSLAC1 I286V A287V and the PdSLAC1 I285V 258

A286V mutant did not carry macroscopic anion currents and that AtSLAC1 V272I V273A 259

displayed a conditional phenotype only, indicates that we have identified a critical position 260

within the selectivity filter in the anion channels’ pore that might result in a meta-stable 261

structure in the mutant AtSLAC1 V272I V273A. Thus, it is tempting to speculate that for proper 262

anion discrimination additional residues are involved. 263

To find a molecular explanation to this discrepancy in monocot-dicot pore residue exchange, 264

we thus asked which channel sites co-evolved with the TMD3 signature. Using a bioinformatics 265

Schäfer et al.

10

co-evolution pipeline (www.evfold.org), we identified residues in AtSLAC1 that co-evolved 266

with the signature motif. Interestingly, the 50 highest scoring hits of co-evolved residues were 267

found exclusively on TMD1 to 3 but not on other parts of the SLAC1 protein (figure 4C, Table 268

S3). To test whether TMD1 to 3 including the remarkable difference in TMD3 between 269

monocot and dicot SLAC1 representatives is essential and sufficient to provide for the monocot 270

nitrate dependency and dicot nitrate independency, we exchanged either only TMD1 and 2 or 271

TMD1, 2 and 3 between AtSLAC1 and HvSLAC1. The resulting chimeras were named 272

AtSLAC1(HvTMD1-2) where AtSLAC1 carries TMD1 and 2 from HvSLAC1, 273

AtSLAC1(HvTMD1-3) where AtSLAC1 carries TMD1-3 from HvSLAC1 and vice versa. 274

When comparing WT SLAC1 channels with the chimeras where only TMD1 and 2 was 275

replaced, we could not document any change in their chord conductance (figure 4D). Only when 276

TMDs 1-3 were exchanged, the nitrate-dependency of HvSLAC1 could be transferred to 277

AtSLAC1 while HvSLAC1 lost its nitrate-dependency when equipped with TMD1-3 of 278

AtSLAC1 (figure 4D). The comparison of rel. open probabilities between AtSLAC1 WT and 279

AtSLAC1(HvTMD1-3) demonstrates that the activation of the chimera is based on a nitrate-280

dependent shift of its rel. PO to negative membrane potentials just like with monocot HvSLAC1 281

WT (figure 4E and F). On the contrary, the chimera HvSLAC1(AtTMD1-3) appeared open 282

even in the absence of nitrate, just like dicot AtSLAC1 WT (figure 4E and F). Thus, the TMD3 283

IA signature from monocots is required and sufficient to convert the dicot SLAC1 from 284

Arabidopsis into a nitrate-gated monocot grass SLAC1 anion channel (figure 4B). In contrast, 285

monocot SLAC1s require the VV motif and in addition backbone residues situated on TMD1 286

and 2 to be converted in a nitrate-insensitive anion channel (figure 4D to F). 287

Schäfer et al.

11

Discussion 288

One of the most striking results to emerge from the experiments described in this paper is that 289

the rates of stomatal opening and closure in barley are much faster than in Arabidopsis (cf. 290

[37]). In the absence of other compensatory factors this is likely to confer a selective advantage 291

on barely in comparison with Arabidopsis. The ability to rapidly adjust stomatal aperture to suit 292

the prevailing environmental conditions is likely to allow barley enhanced control over 293

transpiration, xylem-based nutrient delivery, and photosynthesis that will likely play out in 294

terms of increased competitiveness ([4, 38], and refs. therein). 295

In seeking a mechanistic explanation for the ability of barley stomata to open and close rapidly 296

we focussed first on the flux of the major osmotically active cations and anions. Our results 297

suggest that barley has evolved a stomatal system in which the guard and subsidiary cells 298

behave as a functional unit [4, 37]. For example, during closure while there is loss of K+, Cl- 299

(figure 1C) and very likely NO3- too from the guard cell, it accumulates in the subsidiary cells. 300

In this sense, the subsidiary cells have evolved to play a role as reservoirs or cisterns of 301

osmotically active ions. In grasses stomata move faster than those of dicots because guard cells 302

and subsidiary cells actively regulate turgor and volume in an inverse manner [4, 9]. This way 303

subsidiary cells operate as a source of osmotica that is used by guard cells when they swell, and 304

the stomatal pore opens (figure 1C). 305

A comparative transcriptomic approach of ABA signalling in the cells of the barley stomatal 306

complex revealed the presence of components that were well known from investigations of 307

Arabidopsis guard cells (table S2). These data suggested that, in addition to the evolution of 308

functionally linked and morphologically distinct guard and subsidiary cells, we should look for 309

augmentation of known players in addition to novel elements to explain the rapid movements 310

in barley stomata. 311

312

A tandem amino acid signature in monocot SLAC1 anion channels provide for nitrate 313

dependent gating 314

Here we focussed on the barley SLAC1 anion channel. The regulation of this channel by ABI1 315

and OST1 appeared to be highly conserved between Arabidopsis and barley (figure 2B and C, 316

[14, 15]). The most striking feature to emerge was that gating of the barley guard cell anion 317

channel is controlled by ABA signalling and nitrate (figure 2 and 3). Using a structural biology 318

approach coupled with site-directed mutagenesis, we identified the key residues located in 319

Schäfer et al.

12

TMD3 that are responsible for nitrate gating (figure 4). Interestingly, structure-function 320

investigations with AtSLAH2 also identified Serine 228 (equivalent to V273 in AtSLAC1) in 321

TMD3 as the key residue for the strict nitrate selectivity of the root anion channel [18]. Monocot 322

SLACs could only be converted to nitrate independent dicot-like anion channels when TMD3 323

together with TMD1 and 2 were exchanged (figure 4D to F). This indicates that during SLAC1 324

evolution in monocots, residues on TMD1 and 2 coevolved together with the IA motif in TMD3 325

to form the nitrate gating site (figure 4C, Table S3). Whether and how the intrinsic nitrate sensor 326

in monocot SLAC1 anion channels contribute to the evolutionary success of cereals remain to 327

be shown. However, it is tempting to speculate that SLAC1 anion channels harbouring an 328

intrinsic nitrate sensor might allow the plant to integrate leaf nitrate levels and the 329

velocity/degree of stomatal closure. 330

331

Evolution of the SLAC1 pore properties 332

We and others showed that SLAC1 channels in dicots are permeable to chloride and nitrate and 333

do not require nitrate as gating modifier (this study and [14, 15, 17, 39]). In contrast, monocot 334

SLAC1 channels, such as HvSLAC1, PdSLAC1 and OsSLAC1, require nitrate at the 335

extracellular face of the anion channel pore to gate open, which is reminiscent of the nitrate 336

dependent gating of AtSLAH3 and PttSLAH3 anion channels [17, 31, 40, 41]. Sequence 337

comparisons identified a VV signature in dicot and an IA signature in monocot SLACs that 338

clearly differ between these two evolutionary distinct plant lineages (figure S3 and S4A). 339

Interestingly, similar to monocot SLAC1s, nitrate activated SLAH2/3 anion channels also 340

possess either an IA or an IS motif on TMD3 (figure S3). 341

SLAC1-type anion channels are found in the most basal land plants such as green algae 342

Klebsormidium nitens as well as in the liverwort Marchantia polymorpha [30]. With the 343

emergence of stomata in mosses such as Physcomitrella patens, SLAC1 anion channels co-344

opted the fast ABA-signaling cascade and became OST1-sensitive [30]. figure S3 and S4A 345

shows that the IA motif appeared in moss first and remained largely conserved until the 346

emergence of Arecales (date palm) and Poales lineages that include important grass crops such 347

as rice, maize and barley. In the latter monocots, nitrate-dependent gating is fully functional 348

(this study and [21]). This raises the question of when in evolution SLAC1-type anion channels 349

carrying an IA motive evolved nitrate-dependent gating? 350

To answer this question, we analysed the chord conductance of a set of SLAC1 anion channels 351

derived from evolutionary distinct basal plant lineages including the moss Physcomitrella 352

Schäfer et al.

13

patens, the lycophyte Selaginella moellendorffii, the fern Ceratopteris richardii and the 353

seagrass Zostera marina. Apart from the moss PpSLAC1, all tested basal SLAC1 anion 354

channels appeared equally nitrate insensitive as the dicot SLAC1 anion channels from 355

Arabidopsis, tobacco and tomato (figure S4B, figure 2D). This may indicate that nitrate 356

dependent gating evolved as recently as the emergence of monocot species, although the IA 357

motif on TMD3 is already established in a majority of SLAC-type anion channels of basal plant 358

lineages (figure S3 and S4A). In contrast, following the split between dicots and monocots, the 359

SLAC1s from dicot species lost the IA signature and did not develop a nitrate dependent gating 360

mechanism (figure S4A). 361

To further support that nitrate dependent gating in monocot species evolved after the split 362

between monocots and dicots, we employed a probabilistic approach [42], to infer the most 363

probable core SLAC1 sequence (TMD1 to 10) of the common ancestor from which all extant 364

dicot and monocot SLAC1s evolved (figure S4A). This inferred core sequence was synthesized, 365

equipped with the N- and C-terminus of AtSLAC1 and named Ancestral Slow Anion Channel 366

1 (AncSLAC1, sequence can be found in table S4). Interestingly, AncSLAC1 carried the IA 367

motif on TMD3 just like monocot and basal SLAC1s. Following co-expression with CPK6 in 368

Xenopus leavis oocytes, AncSLAC1 displayed typical S-type anion currents that slowly 369

deactivated at hyperpolarized membrane potentials (figure S4C). In line with SLAC1s from 370

dicots but in contrast to nitrate activated monocot SLAC1 channels, AncSLAC1 mediated 371

macroscopic anion currents in both chloride and nitrate-based media (figure S4B and C) and 372

showed no nitrate dependent gating behavior (figure S4D). Thus, both the predicted common 373

ancestor of dicot and monocot SLAC1 channels AncSLAC1 as well as SLAC1 channels from 374

basal plant lineages were equipped with the IA motif on TMD3 but displayed a largely nitrate 375

independent gating behavior, suggesting that monocots have evolved the nitrate dependent 376

gating mechanism after the split from the dicot species. Future studies will address the question, 377

which backbone sites had to emerge in TMD1 and/or 2 together with the IA motif to form a 378

nitrate-sensitive SLAC1 gate in monocots. 379

Schäfer et al.

14

Acknowledgements: Funded by Bavarian State Ministry of the Environment and Consumer 380

Protection. R.H. and D.G. were supported by the German Research Foundation (DFG) within 381

the CRC/TR166 ‘‘ReceptorLight’’ project B8 and by the King Saud University, Saudi Arabia. 382

M.E.J. is supported by a grant from the Danish Council for Independent Research: DFF–6108-383

00122. We thank Andreas Ruth for Zostera marina plant material and Ingo Dreyer for initial 384

analysis of signatures in SLAC1 isoforms. 385

386

Author contributions: P.A., M.F., J.H., M.E.J., C.L., S.L., T.M., K.v.M. and N.S. performed 387

the research and analysed the data. T.M., P.A., D.G., A.M.H., and R.H. designed the study. 388

T.M., M.E.J., P.A., J.F., D.G., A.M.H., and R.H. wrote the manuscript. 389

390

Declaration of interests 391

The authors declare that there is no competing financial interest. 392

393

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584

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Figure legends 585

Figure 1: ABA induced stomatal closure requires NO3-. Barley stomatal movement 586

measured by infrared gas exchange. Barley stomata were opened in the light (400 μE) at 587

ambient CO2 (400 ppm). (A) Excised leaves were supplied with water and ABA via the 588

transpiration stream. Note, stomatal closure in the presence of ABA was remarkably slow and 589

largely incomplete. (B) Leaves were pre-incubated with 5 mM nitrate. Under this condition 590

leaves started to close their stomata 5 min after ABA application. This process was largely 591

completed within 10 minutes and finished in less than 15 min. Data were normalized to their 592

open value 10 minutes before the application of ABA (indicated by the arrow). n ≥ 5 means ± 593

SE. (C) EDX-analysis of barley stomatal complexes. The respective changes in K- (left) and 594

Cl-contents (right) are shown after the transition from open to closed stomata in guard cells 595

(GC) and subsidiary cells (SC). During stomatal opening potassium and chloride ions shuttle 596

from subsidiary cells to guard cells, while ions move in the opposite direction when stomata 597

close. n = 11, means ± SE. 598

Figure 2. Nitrate-dependent activation of HvSLAC1. See also figure S2. (A) Whole-oocyte 599

currents of Xenopus oocytes expressing HvSLAC1 alone or co-expressing either AtOST1 or 600

AtCPK6 were measured in response to the standard voltage protocol. Currents were recorded 601

in standard buffers containing either 3 mM chloride, 30 mM chloride or 30 mM nitrate. 602

Representative cells of 2 independent experiments with n = 3 oocytes are shown. See also 603

figure S2A. (B) Whole-oocyte currents of oocytes expressing HvSLAC1 equipped with the C-604

terminal half of YFP (HvSLAC1:YC) either expressed alone or together with WT OST1, OST1 605

D140A or WT OST1 and ABI1. Both OST1 versions were fused to the N-terminal half of YFP 606

(OST1:YN, OST1 D140A:YN). Currents were recorded in nitrate-based buffers (30 mM). 607

Representative cells are shown. Co-expression of HvSLAC1 and OST1 or OST1 D140A was 608

confirmed by detection of YFP fluorescence. Quarter of representative oocytes of 2 independent 609

experiments with n = 4 oocytes are shown. (C) Statistical analysis of the steady-state currents 610

at -100 mV derived from the experiment described in (B) (n = 4 experiments, mean ± SD). (D) 611

Chord conductance recorded at a membrane potential of -120 mV of oocytes co-expressing 612

AtOST1 with SLAC1 from different plant species indicated in the figure. Chord conductance 613

was calculated from instantaneous currents recorded in chloride- or nitrate-based buffers (100 614

mM). Chord conductance in nitrate was set to 1 (n ≥ 4 from 2 independent experiments, mean 615

± SD). 616

Schäfer et al.

20

Figure 3: Nitrate activates HvSLAC1 by shifting its rel. open probability to 617

hyperpolarized voltages. See also figure S2. (A) Nitrate-dependence of steady-state currents 618

(ISS) of oocytes co-expressing HvSLAC1 and AtCPK6 are plotted as a function of the applied 619

membrane potential (n = 4 from 2 independent experiments, mean ± SD). (B) The relative open 620

probability (rel. PO) measured in different NO3- concentrations of HvSLAC1/AtCPK6-621

expressing oocytes was plotted against the membrane potential. Data points were fitted with a 622

single Boltzmann equation (solid lines, n = 4 from 2 independent experiments, mean ± SD). 623

(C) Steady-state currents (ISS) of oocytes co-expressing HvSLAC1 and AtCPK6 recorded in the 624

presence of different external chloride concentrations or 30 mM nitrate (n = 4from 2 625

independent experiments, mean ± SD). (D) The relative open probability (rel. PO) of HvSLAC1 626

in the presence of different Cl--concentrations or 30 mM NO3- was plotted against the 627

membrane potential. Data points were fitted with a single Boltzmann equation (solid lines, n = 628

4 from 2 independent experiments, mean ± SD). (E) The half-maximal PO (V1/2) calculated from 629

the data in (B) was plotted against the nitrate concentration. A Michaelis-Menten equation was 630

used to calculate a K0.5 of 10.9 mM NO3- (n = 4, mean ± SD). (F) Steady-state currents of 631

HvSLAC1 and AtCPK6 co-expressing oocytes in the presence of different Cl-/NO3--ratios were 632

plotted against the applied voltage (n = 4 from 2 independent experiments, mean ± SD). (G) 633

The relative open probability (rel. PO) of HvSLAC1 in different Cl-/NO3--ratios was plotted 634

against the membrane potential. Data points were fitted with a single Boltzmann equation (solid 635

lines, n = 4 experiments, mean ± SD). 636

Figure 4: IA-motif on TMD3 coevolved with residues on TMD1 and 2 to provide monocot 637

SLAC1 anion channels with a nitrate-depending gating mechanism. See also figure S3 and 638

S4 as well as table S3 and S4. (A) Frequency loges of transmembrane three (TMD3) from 639

SLAC1 anion channels of different monocot or dicot species. The respective sequence 640

alignment is shown in figure S3. The most prominent difference (IA motif in monocots vs. 641

VV/IV motif in dicots) is marked with a red box. (B) Chord conductance at -120 mV of 642

AtSLAC1 WT and AtSLAC1 V272I V273A compared to HvSLAC1 WT and HvSLAC1 I286V 643

A287V or PdSLAC1 WT and PdSLAC1 I285V A286V. All channels and mutants thereof were 644

co-expressed with CPK6. Currents were recorded in nitrate- or chloride-based buffers. (n = 4 645

from 2 independent experiments, mean ± SD). Note, the phenotypes of HvSLAC1 and 646

AtSLAC1 anion channels were highly reproducible showing nitrate-independent gating 647

properties of AtSLAC1 and nitrate-dependent gating of HvSLAC1 expressing oocytes. In 648

contrast, with the mutant AtSLAC1 V272I V273A we observed a conditional phenotype 649

strongly dependent on the investigated oocyte batch. In 30 % of the tested oocyte batches the 650

Schäfer et al.

21

mutations in the selectivity signature converted AtSLAC1 into a HvSLAC1-type nitrate-651

dependent anion channel (these data are shown in this study) whereas the remaining oocyte 652

batches revealed a AtSLAC1 WT behaviour. (C) Evolutionary coupling analysis. The top 50 653

amino acid residues (see also table S3) that showed evolutionary coupling to AtSLAC1-V272 654

and V273 (purple spheres) were highlighted in red on previously generated homology models 655

[18, 36] using VMD [43]. Note, some of the highlighted residues co-evolved with both V272 656

and V273. The sphere size of co-evolved residues does not relate to the evolutionary coupling 657

strength but reflects the side chain size. TMD1 is depicted in dark grey, TMD2 in green and 658

TMD3 in light grey. The remaining TMDs are shown in transparent orange. (D) Chord 659

conductance of oocytes co-expressing AtCPK6 with either AtSLAC1, HvSLAC1 or one of the 660

indicated chimeras. Currents were recorded in nitrate or chloride-based buffers. Chord 661

conductance for nitrate was set to 1 (n = 4 from 2 independent experiments, mean ± SD). See 662

also Figure S4B. (E) and (F) Relative open probability (rel. PO) of (E) AtSLAC1 and the 663

chimera AtSLAC1(HvTMD1-3) or (F) HvSLAC1 and HvSLAC1(AtTMD1-3) in the presence 664

of 30 mM chloride or nitrate (n=4 from 2 independent experiments, mean ± SD). 665

666

Schäfer et al.

22

STAR Methods 667

CONTACT FOR REAGENT AND RESOURCE SHARING 668

Further information and requests for resources and reagents should be directed to and will be 669

fulfilled by the Lead Contact, Dietmar Geiger (geiger@botanik.uni-wuerzburg.de). 670

671

EXPERIMENTAL MODEL AND SUBJECT DETAILS 672

Plant material and growth conditions 673

Barley (Hordeum vulgare cv. Barke) seeds were provided by a commercial supplier (Saatzucht 674

J. Breun GmbH & Co. KG) and cultivated at 22/16 °C and 50 ± 5% RH at a 12/12h day/night 675

cycle and a photon flux density of 500 μmol m-2 sec-1 white light (Philips Master T Green 676

Powers, 400 W). 677

Xenopus oocyte preparation 678

Investigations on SLAC1 anion channels were performed in oocytes of the African clawfrog 679

Xenopus laevis. Permission for keeping Xenopus exists at the Julius-von-Sachs Institute and is 680

registered at the government of Lower Franconia (reference number 70/14). Mature female 681

Xenopus laevis frogs (healthy, non-immunized and not involved in any previous procedures) 682

were kept at 20 °C at a 12/12h day/night cycle in dark grey 96 litres tanks (5 frogs/tank). Frogs 683

were fed twice a week with floating trout food (Fisch-Fit Mast 45/7 2mm, Interquell GmbH, 684

Wehringen, Germany). Tanks are equipped with 30 cm long PVC pipes with a diameter of 685

around 10 cm. These pipes are used as hiding places for the frogs. The water is continuously 686

circulated and filtered by a small aquarium pump. For oocyte isolation, mature female X. laevis 687

frogs were anesthetized by immersion in water containing 0.1% 3-aminobenzoic acid ethyl 688

ester. Following partial ovariectomy, stage V or VI oocytes were treated with 0.14 mg/ml 689

collagenase I in Ca2+-free ND96 buffer (10 mM HEPES pH 7.4, 96 mM NaCl, 2 mM KCl, 1 690

mM MgCl2,) for 1.5 h. Subsequently, oocytes were washed with Ca2+-free ND96 buffer and 691

kept at 16 °C in ND96 solution (10 mM HEPES pH 7.4, 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 692

1mM CaCl2) containing 50mg/l gentamycin. For oocyte BiFC and electrophysiological 693

experiments 10 ng of each cRNA was injected into selected oocytes. Oocytes were incubated 694

for 2 days at 16 °C in ND96 solution containing gentamycin. 695

696

METHODS DETAILS 697

Field Code Changed

Schäfer et al.

23

RNA sequencing 698

Epidermal peels were collected from the abaxial side of 8 to 10-day-old leaves. To prepare 699

isolated epidermal peels [5], leaves were cut from the plant and bent over the forefinger with 700

the adaxial surface facing upward. A shallow cut was made with a sharp razor blade horizontally 701

across the leaf and a flap of leaf tissue lifted with a razor, leaving the lower epidermis intact. 702

The leaf tissue was removed from the epidermis with forceps. RNA was extracted from a total 703

of 20 epidermal peels per sample using the NucleoSpin® RNA Plant Kit (Macherey-704

Nagel,Drueren, Germany). RNA isolation from whole leaves was performed similarly. 705

The extracted RNA was treated with RNase-free DNase (New England Biolabs, Ipswich, MA, 706

USA). Quality control measurements were performed on a 2100 Bioanalyzer (Agilent, Santa 707

Clara, CA, USA) and the concentration was determined using a Nanodrop ND-1000 708

spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Following RNA 709

isolation, we sequenced RNA of one sample, each to get a first overview of genes in barley 710

guard cell complexes that are known to be involved in stomatal movements. In addition, we 711

were thus able to obtain the sequence information for cloning selected transporters and 712

channels. Libraries were prepared with the TruSeq RNA Sample Prep Kit v2 (Illumina, San 713

Diego, CA, USA) using 1 µg of RNA and sequenced on a HiSeq 3000 (Illumina) resulting in a 714

sequence depth of 35 million paired-end reads (2x 150bp). 715

RNA-seq data analysis 716

Sequencing adaptors were initially removed, and the overall high quality of the remaining reads 717

was confirmed using FastQC (FASTQC v0.10.1, Andrews: 718

http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (average Phred quality score of 719

>30 across all bases at each position in the FastQ files). Subsequently, reads were aligned to 720

the barley reference assembly [44] using Hisat2 (hisat2-2.0.3-beta) [45] by applying default 721

settings for paired-end data. The featureCounts function of the subread-1.4.6 package [46] was 722

used to generate counts for high-confidence genes of barley [44]. Only uniquely mapped read 723

pairs were counted. Data normalization was performed by calculating TPM (Transcripts Per 724

Kilobase Million) [47] values. 725

Functional annotation of barley 726

The functional annotation of the barley genes was provided by Mascher et al. (2017 [44]). Best 727

hits of a BLASTP [48] alignment of barley high-confidence protein sequences against the A. 728

Schäfer et al.

24

thaliana protein sequences (TAIR10, [49]) were used to assign A. thaliana genes and the 729

corresponding MapMan categories [50] to barley genes. 730

Gas exchange experiments 731

For gas exchange measurements [21], we used detached leaves of 8 to 10-day-old Hordeum 732

vulgare cv. Barke. The leaves were cut under water to avoid xylem embolism and immediately 733

placed in deionized water or 5 mM KNO3 and kept there for the whole measurement period. 734

The effect of ABA application with or without KNO3 on the transpirational water loss was 735

measured at a photon flux density of 500 µmol m-2s-1. After stabilization of the transpiration 736

ABA with a final concentration of 25 µM was fed into the water reservoir containing either 737

deionized water or 5 mM KNO3. Transpirational water loss was measured under constant 738

conditions: air humidity of 52.5 %, temperature of 20 °C, and a photon flux density of 500 µmol 739

m-2 s-1. 740

qPCR 741

Quantitative PCR (qPCR) experiments were performed with samples taken from whole leaves 742

epidermal peels and highly guard cell enriched tissue. Epidermal peels from 12-day-old Barley 743

(cv. Barke) leaves were isolated according to [5]. Thereby only the guard cell subsidiary cell 744

complex survives. For guard cell samples we used the “blender method” on epidermal peels 745

with mature, intact guard cells to mechanically and selectively destroy the subsidiary cells while 746

keeping guard cells alive. Guard cell were enriched within 8 minutes by successive blender 747

cycles (45 seconds each) in ice-cold deionized water with additional crushed ice and filtered 748

through a 210-µm nylon mash. After two rounds of blending, the remaining light green 749

epidermal fraction was further processed. Neutral red staining indicated that at least 90% of the 750

viable cells in the preparations were guard cells. Total RNA from at least three individual 751

biological replicates was prepared using the NucleoSpin® RNA Plant Kit (Macherey Nagel, 752

Drueren, Germany) and stored for subsequent microarray hybridizations or qPCR. 753

For qPCR potential DNA contamination was removed from total RNA by treatment with 754

RNase-Free DNase I (Thermo Scientific, Waltham MA) according to the manufacturer’s 755

protocol. First-strand cDNA was prepared using 2.5 µg RNA with the M-MLV-RT kit 756

(Promega, Mannheim, Germany). First-strand cDNA samples were 20-fold diluted in water and 757

subjected to qPCR using a Mastercycler® ep Realplex2S (Eppendorf) with the ABsolute SYBR 758

Capillary Mix (Thermo Scientific, Waltham MA) in 20 µl reaction volumes. Primers used (TIB 759

MOLBIOL, Germany) have been designed according to the sequences from the RNA-seq 760

analyses and validated prior to qPCR. All primers were chosen to amplify fragments not 761

Schäfer et al.

25

exceeding 500 base pairs. Each transcript was quantified using individual standards. To enable 762

detection of contaminating genomic DNA, PCR was performed with the same RNA as template 763

that was used for cDNA synthesis. Transcripts were each normalized to 10.000 molecules of 764

barley actin4/1. These barley actin fragments, used as house-keeping genes, were homologous 765

to actins 2 and 8 constitutively expressed in most Arabidopsis tissues (for details see [51, 52]. 766

All kits were used according to the manufacturer’s protocols. The primers are listed in the Key 767

Resources Table. 768

Energy dispersive X-ray analysis (EDXA) 769

Leaf samples with open and closed stomata were prepared using the gas exchange setup. Cut 770

leaves were either treated with opening conditions (500 µmol m-2 s-1 light, 0 ppm CO2) or 771

closing conditions (darkness, 1000 ppm CO2) until the transpirational water loss stabilized. The 772

samples were then immediately frozen in liquid nitrogen and lyophilized over a period of 3 days 773

in an ice condenser (Alpha 1-2, Christ GmbH, Germany) under vacuum (R25, Vacuubrand 774

GmbH & CO. KG, Germany) at -55 °C. After the following freeze-drying process leaves were 775

coated with carbon before being examined by a scanning electron microscope (SEM, S-520 776

Hitachi, Tokyo, Japan) equipped with an energy dispersive X-ray device (EDX eumex Si(Li)-777

detector, EUMEX GV, Mainz, Germany). Single-point measurements on guard cells as well as 778

on subsidiary cells were performed at 10 keV excitation energy, which excites a measurement 779

area of < 2 µm in diameter. Element concentration provided by the analysis data represents the 780

atomic ratio of the analysed ions in percent. 781

Cloning and cRNA synthesis 782

The complementary DNAs (cDNAs) of various SLAC1 anion channels, AtSLAH2, AtCPK6, 783

AtABI1 and AtOST1 were cloned into oocyte expression vectors or BiFC expression vectors 784

(both are based on pGEM vectors), by an advanced uracil-excision-based cloning technique as 785

described by [53]. Site-directed mutations were introduced by means of a modified USER 786

fusion method as described by [54, 55]. In brief, the coding sequence of the respective anion 787

channel or kinase within an oocyte expression vector (based on pNBIu vectors, see KEY 788

RESOURCES TABLE) was used as a template for USER mutagenesis. Overlapping primer 789

pairs (overlap covering 8 to 14 bp including the mutagenesis site, see Table S5) were designed 790

[53]. PCR conditions were essentially as described by Nørholm et al (2010, [55]) using PfuX7 791

polymerase. PCR products were treated with the USER enzyme (New England Biolabs, 792

Ipswich, MA, USA) to remove the uracil residues, generating single-stranded overlapping ends. 793

Following uracil excision, recirculation of the plasmid was performed at 37°C for 30 minutes 794

Schäfer et al.

26

followed by 30 minutes at room temperature, and then constructs were immediately 795

transformed into chemical competent Escherichia coli cells (XL1-Blue MRF’). All mutants 796

were verified by sequencing. [54]. The cDNA of Arabidopsis/barley chimeras was also cloned 797

into oocyte expression vectors using a combination of the advanced uracil-excision-based 798

cloning technique and the USER fusion technique [53, 56]. Primers are listed in table S5. For 799

functional analysis, complementary RNA (cRNA) was prepared with the AmpliCap-Max T7 800

High Yield Message Maker Kit (Cellscript, Madison, WI, USA). Oocyte preparation and cRNA 801

injection is described in Experimental Model and Subject Details. 802

Protein-protein interaction studies (BiFC) 803

For documentation of the oocyte BiFC results, pictures were taken with a Leica SP5 confocal 804

laser scanning microscope (Leica Microsystems CMS GmbH, Mannheim, Germany) equipped 805

with multiphoton laser of the Mai Tai-Series (Spectra Physics, Santa Clara, USA) and a Leica 806

HCX IRAPO L25×/0.95W objective. 807

Oocyte recordings 808

In double-electrode voltage-clamp studies, oocytes were perfused with Tris/Mes-based buffers. 809

The standard solution contained 10 mM Tris/Mes (pH 5.6), 1 mM Ca(gluconate)2, 1 mM 810

Mg(gluconate)2, 1 mM LaCl3 and 100 mM NaCl, NaNO3 or Na(gluconate). To balance the ionic 811

strength, we compensated for changes in the nitrate or chloride concentration with 812

Na(gluconate). Solutions for anion selectivity measurements were composed of 50 mM malate-813

, sulphate2-, Cl−, NO3− or gluconate−, 1 mM Ca(gluconate)2; 1 mM Mg(gluconate)2; and 10 mM 814

Tris/Mes (pH 7.5). Osmolality was adjusted to 220 mosmol/kg with D-sorbitol. For recording 815

representative current traces, steady-state currents (ISS) and for calculating the voltage 816

dependent relative open probability (rel. PO) standard voltage protocol was as follows: Starting 817

from a holding potential (VH) of 0 mV, single-voltage pulses were applied in 20 mV decrements 818

from +40 to −200 mV. Rel. PO was calculated from a −120 mV voltage pulse following the test 819

pulses of the standard voltage protocol by fitting the experimental data points with a Boltzmann 820

equation [31] of the form: rel. PO = offset + 1 / (1 + exp (V1/2 – Vm) / z), where V1/2 is the half 821

maximal activation voltage, Vm is the membrane potential and z is the slope of the Boltzmann 822

function. The currents were normalized to the saturation value of the calculated Boltzmann 823

distribution. Instantaneous currents (Iinst) were extracted immediately after the voltage jump 824

from the holding potential of 0 mV to 50 ms test pulses ranging from +70 to −150 mV. The 825

reversal potentials (Vrev) used for the calculation of the rel. permeability were recorded in the 826

current-clamp mode [18]. For determination of Vrev for the respective anion, oocytes were 827

Schäfer et al.

27

preincubated in 50 mM NO3- to gain full activity of the channel. The relative permeability was 828

calculated as described in [36] using the following equation: 829

𝑃𝑋

𝑃𝑁𝑂3=

[𝑁𝑂3−]𝑜

[𝑋−]𝑜𝑒(𝐸𝑋−𝐸𝑁𝑂3)𝐹

𝑅𝑇 for monovalent anions and 𝑃𝑋

𝑃𝑁𝑂3=

[𝑁𝑂3−]𝑜

4[𝑋2−]𝑜𝑒(𝐸𝑋−𝐸𝑁𝑂3)𝐹

𝑅𝑇 (𝑒−𝐸𝑋𝐹

𝑅𝑇 + 1) 830

for anions differing in valence (divalent and monovalent). [NO3−]o is the external concentration 831

of the control (nitrate-based) solution and [X−]o is the external concentration of the test anion. 832

ENO3 is the reversal potential with nitrate and EX is the reversal potential for the external test 833

anion. F and R are the Faraday and gas constants, respectively, and T is the absolute 834

temperature. 835

To calculate the chord conductance, the reversal potential (Vrev) was determined by fitting the 836

instantaneous currents in chloride- and nitrate-containing standard buffers with a linear 837

function. Using the instantaneous currents at –120 mV, the chord conductance could be 838

calculated with the equation ganion = Ianion / (V - Vrev) [31]. 839

Evolutionary coupling analysis 840

EVfold/EVcouplings (www.evfold.org) [57, 58], a publicly available bioinformatics server, 841

was used to predict evolutionary couplings of the amino acid residues V272 an V273 in 842

AtSLAC1. We used the default transmembrane protein settings and the DI setting as the 843

coupling scoring function. Multiple sequence alignment was done with default settings and 844

resulted in 468 sequences with a e-value cut off of -3. The top 50 amino acid residues (see table 845

S3) that showed evolutionary coupling to AtSLAC1-V272 and V273 were highlighted on 846

previously generated homology models [36] using VMD [43]. 847

Frequency logos of TMD1 to 3 848

Selected SLAC1 homologs were identified by BLASTP (Sequences can be found in table S4). 849

Sequences were aligned using MUSCLE [59] with a gap open penalty of -2.9, gap extend of 0 850

and hydrophobicity multiplier of 5. Transmembrane helix 1, 2 and 3 were identified in the 851

homology model of AtSLAC1 [18] using chimera [60]. Frequency logos were created based on 852

alignments of transmembrane helix 1, 2 and 3 using the weblogo program [61]. Jalview [62] 853

was used to visualize transmembrane helix 1, 2 and 3 alignments. 854

Alignment, phylogenetic analysis and inference of ancestral SLAC1 855

Alignment 856

Field Code Changed

Schäfer et al.

28

Selected SLAC1 homologs were identified by BLASTP. Sequences were aligned using 857

MUSCLE [59] with a gap open penalty of -2.9, gap extend of 0 and hydrophobicity multiplier 858

of 5. Gaps introduced by parts of the sequence supported by 4 or less genes were trimmed. 859

Phylogenetic analysis 860

MrBayes 3.2.6 [63] was used to infer the Bayesian phylogenetic tree as previously described 861

[64]. Briefly, Prottest v3.4.2 [65] identified the appropriate LG based phylogenetic model as 862

LG+I+G that use a general amino acid replacement matrix [66] with a proportion of invariable 863

sites (+I) [67] that use a gamma distribution for modelling the rate heterogeneity (+G) [68]. 864

Bayesian inference trees were calculated until convergence was reached (“average standard 865

deviation of split frequencies” <0.01). The temperature heating parameter was set to 0.05 866

(temp=0.05) to increase the chain swap acceptance rates, this reduce the chance of Markov 867

chains getting stuck at local high-probability peaks. Burn-in was set to 25% (burninfrac=0.25) 868

and the number of Markov chains was set to 8 (nchains=8). 869

RAxML 870

The maximum likelihood phylogenetic tree was inferred using RAxML 8.2.9 as previously 871

described [64]. Briefly, Prottest v3.4.2 [65] identified LG+I+G as the best phylogenetic model. 872

1000 bootstrap replicate searches were performed, and the bootstrap values were portrayed on 873

the MrBayes generated consensus tree when MrBayes values were below 0.95. SLAC1 from 874

MP was used as the out-group. All analyses were run in MPI via the CIPRES SCIENCE 875

GATEWAY [69] at the San Diego Supercomputer Center (SDSC). Trees were visualized in 876

figtree (http://tree.bio.ed.ac.uk/software/figtree/) and annotated with Adobe Illustrator. 877

Inference of ancestral sequence 878

The topology of the phylogenetic tree inferred by MrBayes and RAxML were identical and we 879

used this multiple sequence alignment and the phylogenetic tree as input for inference of the 880

ancestral sequence of the last common ancestor of monocots and dicots. Probabilities for the 881

ancestral sequence at the split between monocots and dicots (figure S4A) was calculated using 882

the LG model of substitution by FastML [42]. The top 100 most likely sequences showed a log 883

likelihood difference of only 0.19 which suggests that sequence #1 and #100 are almost as likely 884

to be true. The N- and C-terminus of SLAC1 has activating/regulatory roles and thus we, 885

respectively, substituted the residues 1-182 and 514-556 from the AncSLAC1 N- and C-886

terminal residues with the corresponding residues from AtSLAC1 (for sequence information 887

Field Code Changed

Schäfer et al.

29

see table S4). We resurrected AncSLAC1 by GeneStrand synthesis (Eurofins Medigenomix 888

GmbH, Campus Ebersberg, Germany). 889

890

QUANTIFICATION AND STATISTICAL ANALYSES 891

All experiment was performed at least two times (independent experiments). Sample size, n, 892

and statistical details (mean ± standard error, SE or standard deviation, SD) for each experiment 893

are given in the figure legends. Statistical significances based on one-way ANOVA. For 894

statistical analysis the software Igor Pro7 (waveMetrics, Inc., Lake Oswego, Oregon, USA), 895

Excel (Microsoft Corp. Redmond, Washington, USA) was used. 896

897

DATA AND SOFTWARE AVAILABILITY 898

Raw RNA-seq sequence reads are available at the European Nucleotide Archive with accession 899

number ArrayExpress accession E-MTAB-5877. 900

Accession numbers: HvSLAC1 (Hordeum vulgare cultivar Barke); AtSLAC1 (Arabidopsis 901

thaliana Col-0) At1g12480; OsSLAC1 (Oryza sativa Japonica Group) XP_015636891; 902

SlSLAC1 (Solanum lycopersicum) XP_004245686; NtSLAC1 (Nicotiana tabacum) 903

XP_016515379; ZomSLAC1 (Zostera marina) KMZ58505; PdSLAC1 (Phoenix dactylifera) 904

XP_008780343.1; PpSLAC1 (Physcomitrella patens) PNR63146.1; CrSLAC1a (Ceratopteris 905

richardii) KT238910; SmSLAC1b (Selaginella moellendorffii) KU556809; AtOST1 906

(Arabidopsis thaliana Col-0) At4g33950; AtCPK6 (Arabidopsis thaliana Col-0) At2g17290; 907

AtABI1 (Arabidopsis thaliana Col-0) At4g26080. 908

Software and algorithms used in this study are listed in the KEY RESOURCES TABLE. In 909

addition, for graph preparations and statistical analysis the software Igor Pro7 (waveMetrics, 910

Inc., Lake Oswego, Oregon, USA), Excel (Microsoft Corp. Redmond, Washington, USA), 911

Adobe Illustrator (Adobe Systems Incorporated, San Jose, California, USA) and CorelDRAW 912

(Corel Corporation, Ottawa, Ontario, Canada) was used. 913

Schäfer et al.

30

Supplemental tables 914

Table S1: Differentially expressed genes (DEG) in guard cell complexes (related to figure 915

S1). 916

Table S2: Selection of transcripts that are involved in stomatal movement (related to 917

figure S1). 918

Table S3: The top 50 amino acid residues that showed evolutionary coupling (EC) to 919

AtSLAC1 V272 and V273 are shown here (related to figure 4). 920

Table S4: Gene names, species and SLAC1 amino acid sequences that were used to build 921

alignments, frequency logos and phylogenetic trees (related to figure 4, figure S3 and S4). 922

Table S5: Oligos used in this study (related to figure S1 and METHODS DETAILS). 923